Vitamin D and intestinal cell membranes

Biochimica et Biophysica Acta, 694 (1982) 3 0 7 - 3 2 7

307

Elsevier Biomedical Press

BBA 85234

VITAMIN

D AND

INTESTINAL

I L K A N E M E R E and A N T H O N Y

CELL

MEMBRANES

W. N O R M A N *

Department of Biochemistry, University of California, Riverside, CA 92521 (U.S.A.) ( A c c e p t e d April 27th, 1982)

Contents I.

Introduction

.............................................................................

II.

Brush borders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. G l y c o c a l y x . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. P h o s p h o l i p i d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. E n z y m e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. G u a n y l a t e cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. A l k a l i n e p h o s p h a t a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Leucine a m i n o p e p t i d a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. M a l t a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e. Sucrase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. C a l c i u m - b i n d i n g p r o t e i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. O t h e r p r o t e i n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. C y t o s k e l e t a l proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

308 309 309 310 310 311 312 312 312 312 313 314 314 314 315 315

I l L I n t r a c e l l u l a r organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M i t o c h o n d r i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. G o l g i vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. L y s o s o m e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

316 316 317 317 318

IV. Basal lateral m e m b r a n e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319

V.

320 320 322

Mechanisms ............................................................................. A. Protein carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Vesicular carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements ............................................................................

324

References

324

* T o w h o m c o r r e s p o n d e n c e s h o u l d be addressed. 0 3 0 4 - 4 1 5 7 / 8 2 / 0 0 0 0 - 0 0 0 0 / $ 0 2 . 7 5 © 1982 Elsevier Biomedical Press

308 1. Introduction

The biochemical mechanisms involved in regulating the intracellular ionic environment, as well as those associated with the transcellular movement of ions to effect net transport, have been the subject of intensive investigation for several decades. Studies on calcium uptake across the intestine have constituted a significant proportion of the attention devoted to ionic fluxes, as this system combines the regulation of a cation that is crucial to many cellular events and hormonal modulation of transport. The major hormonal stimulus for increased calcium transport across the intestine is 1,25-dihydroxycholecalciferol [I,25(OH)2D3], a metabolite of vitamin D 3. Potential regulatory sites for 1,25(OH)2D3-dependent calcium transport include enhanced uptake of the cation from the lumen across the microvillar or brush border membrane, increased efficiency of translocation across the cell, and an elevated extrusion rate across the basal lateral membrane into the blood. A detailed examination of specific membraneassociated regulatory sites involved in intestinal calcium transport was not feasible until the metabolic pathway was discovered for the conversion of the parent vitamin D into 1,25(OH)2D 3. The first vitamin D metabolite was discovered in 1966 [1], and subsequently characterized as 25-hydroxycholecalciferol [25(OH)D3] [2]. H o w e v e r , 25(OH)D 3 was soon found to be substrate for further hydroxylation to yield an even more polar product, 1,25(OH)2D 3. This metabolite was independently isolated from the intestine by Norman's laboratory [3,4], Lawson's laboratory [5,6] and DeLuca's laboratory [7]. Within a relatively short span of years, it was reported that the 1-hydroxylation of 25(OH)D 3 occurred in the kidney [8-10], that the intestinal epithelium contains specific receptors for 1,25(OH)2D 3 in cytosol fractions [1115] as well as in nuclear chromatin [3,11,16-18], and that transfer from the cytoplasmic to nuclear compartment [ 11] is a temperature-dependent process [13]. These clear indications that vitamin D 3 metabolism results in the production of a steroid hormone were complimented by the finding that the vitamin D-dependent calcium-binding protein present in the intestine [19], is a specific gene product induced by 1,25(OH)2D 3 [20,21]. A large

amount of circumstantial evidence supported the view that calcium-binding protein was intimately involved in various aspects of vitamin D-mediated intestinal calcium transport [22,23]. Under normal circumstances, calcium-binding protein contributes 0.5-1.5% of the total protein of the intestinal cell. The rapid progress in establishing the hormonal properties of 1,25(OH)2D 3 that occurred between the late 1960s and mid-1970s, suggested that a significant portion of the molecular mechanism of vitamin D-dependent calcium transport would be elucidated within a few years. However, this optimistic outlook was dispelled by the application of radioimmunoassay techniques for calcium-binding protein in a study to determine the onset and time-course of stimulated intestinal calcium transport and the appearance of calcium-binding protein, after administration of 1,25(OH)2D 3 to vitamin D-deficient chicks. As reported by Spencer et al. [24], there was a clear 1,25(OH)2 D3-mediated stimulation of calcium transport which occurred prior to the appearance of detectable levels of calcium-binding protein or even RNA polymerase activity [25]. Further major discrepancies arose between the classical model of steroid hormone action and vitamin D-mediated intestinal calcium transport when Bikle et al. [26] observed that inhibition of de novo RNA or protein synthesis failed to abolish hormonally stimulated uptake and transport of the cation. These findings were subsequently supported by the report of Rasmussen et al. [27]. Also, the tissue localization of calcium-binding protein was found not to be restricted to tissues heavily committed to transcellular calcium transport, such as the intestine and kidney. Significant levels of vitamin D-dependent calcium-binding protein were found in the pancreas [28] and brain [29,30]. The evidence that protein synthesis is not an obligatory step in initiating vitamin D-dependent calcium transport has stimulated an increasing number of studies directed toward assessing alterations in membrane properties that are mediated by vitamin D. The results of some of these studies are presented below under the divisions of membrane locale, i.e., brush border, intracellular, and basal lateral, with further subdivisions into membrane components. These subdivisions are somewhat

309

artifactual in view of cellular continuity and integration, and thus, engender a certain degree of overlap. II. Brush borders

At the subcellular level, vitamin D-dependent alterations in the properties of intestinal epithelial brush borders comprise the majority of studies undertaken in the field of hormonally-modulated transport. Much evidence has been accumulated suggesting that brush border membranes are the chief site of action of a vitamin D-mediated increase in calcium uptake [31-37]. As a specialized region of the plasma membrane, brush borders are amenable to isolation in high yield, e.g., 50% of the starting material [38], with 10-50-fold purification factors for the marker enzyme activities sucrase and leucine aminopeptidase, respectively [38,39 *]. Brush borders can also be subjected to treatments that disrupt associated cytoskeletal components to yield brush border membranes further enriched for marker enzyme activities [38,40-43] and capable of forming sealed, osmotically active vesicles [27,39,41,43,44].

IIA. Glycocalyx Although the mucin coat is largely absent in preparations of isolated brush border membranes, the intimate association of the glycocalyx with the apical epithelial membrane in vivo warrants its inclusion in this review. The available literature concerning the contribution of the glycocalyx to calcium absorption is scant [45-48], although recent evidence suggests that the mucous coating (comprised of mucoproteins, glycoproteins and glycolipids (cf. Ref. 49)) does indeed constitute a major diffusional barrier to solutes as small as hydrogen ions (cf. Ref. 49). In 1963, Kashiwa and Atkinson [47] reported that the greatest concentration of calcium in the rat ileum, as judged by specific cytochemical staining procedures, was associated with goblet cell mucin. These findings were verified by Warner and Coleman in 1975 by electron probe microanalyses [48]. In studies on the effect of vitamin D status on calcium distribution in rat intestine, these * Nemere, S., unpublished data.

same authors reported finding sequestered calcium within goblet cells in preparations from vitamin D-deficient rats and that such localizations were increased in intestinal segments from vitamin Dtreated animals [48]. However, the authors also noted that increased goblet cell calcium localizations coincided with the regions of greatest calcium absorption, i.e., the distal intestine, thereby apparently dissociating goblet cell mucin from vitamin D-dependent absorption of calcium in the proximal intestine [48]. Perhaps the strongest evidence for an association between the intestinal mucous coat and vitamin D-dependent calcium transport was reported by Taylor and Wasserman [45]. Use of an immunofluorescent antibody technique allowed visualization of calcium-binding protein in several locations, mainly within goblet cells and in association with the brush border surface coat [45]. Subsequent studies by Morrissey et al. [50] contradicted the existence of extracellular calciumbinding protein because of redistribution artifacts that occurred during tissue preparation for immunocytochemical localization of the protein [51,52]. However, the controversy may have been revived by the recent report of Galvanovsky et al. [46]. In order to minimize redistribution artifacts generated by preparative procedures, these workers made a replica of chick duodenum by filling an intestinal segment with agar kept at 42°C. After cooling and careful removal of the solidified agar cylinder, biochemical and electron microscopic analyses indicated that the agar replica had removed the glycocalyx above the microvilli, but did not compromise the integrity of the mucosal cells nor that of the intermicrovillar glycocalyx. Application of this technique to duodenal segments from vitamin D-treated and deficient chicks revealed calcium-binding protein was detectable in the removed glycocalyx of replete birds, but not deficient ones. Nevertheless, conclusive evidence for the involvement of glycocalyx-localized calcium-binding protein in vitamin D-mediated calcium transport has not been presented to date, although a mechanism for such a role has been postulated [53]. It cannot be ruled out, however, that reuptake of lumenal calcium-binding protein primarily reflects a mechanism for conservation of proteins lost from senescent or injured cells.

310

liB. Lipids IIB-1. Cholesterol Perhaps the first indication that vitamin D could stimulate calcium uptake by mediating alterations in the lipid bilayer of brush border membranes came from studies using the polyene antibiotic filipin. Application of filipin to the mucosal surface of intestinal segments in vitro was found to selectively stimulate the uptake and transport of calcium, but not other ions, in ilia removed from rachitic chicks [54,55]. In comparable preparations from vitamin D-replete birds, filipin treatment in vitro augmented calcium uptake, but not net transport [54,55]. These effects, however, could not be reproduced by exposure of the mucosa to filipin in vivo (Norman, unpublished data). One explanation for the disagreement between the observed effects of filipin treatment in vivo and in vitro is the integrity of the intestinal mucin coat (see above). In the experiments relying on filipin treatment in vitro, the selected intestinal segments were removed from chicks that had been killed by decapitation [54,55] and at least partially exsanguinated. The ensuing tissue anoxia has been observed in the rat to result in autolysis of intestinal cells with concomitant release of hydrolytic enzymes [56] including glycosidases. The occurrence of sublethal autolysis in dead chicks could compromise the integrity of the mucin coat, thereby allowing access of filipin to brush border membranes, in contrast to administration of the polyene antibiotic in vivo. Such considerations notwithstanding, the stimulation of calcium uptake and transport by filipin treatment of rachitic chick intestine in vitro, suggested that the necessary uptake components were present but inactive in the brush borders. Additional investigations of the interaction of filipin with synthetic membranes had led to the discovery that the polyene antibiotic selectively binds to cholesterol [57]. Since cholesterol levels represent a larger lipid component of brush border membranes than of basal lateral membranes ([42] cf. Ref. 58) a potential mechanism for vitamin Denhanced permeability of brush border membranes is one in which 1,25(OH)2D 3 sequesters cholesterol molecules in the lipid membrane, resulting in the concomitant formation of less

ordered or more fluid lipid microdomains. The increase in bilayer fluidity would then allow the protein molecule(s) of the calcium carrier to assume a more active configuration. However, the experimental tests of a mechanism involving a direct interaction between vitamin D and lipid bilayers have not yielded conclusive results. For example, incorporation of vitamin D3, 25(OH)D3, or filipin in cholesterol-containing black films decreased the resistance of the model membranes, and conferred a degree of cation selectivity to the bilayers [59] but no comparable experiments have been conducted with 1,25(OH)zD 3. Electron spin resonance (ESR) spectroscopy of a variety of lipid probes has also failed to detect altered bilayer fluidity in brush border membranes isolated from chicks treated with 1,25(OH)2D 3 in vivo, relative to preparations from rachitic chicks [42]. Nor has vitamin D status been found to alter the cholesterol: phospholipid ratios in brush borders [42,55]. An indication that the studies using physical techniques (see above) have limited sensitivity, comes from additional observations related to the effects of filipin. Electron microscopic analyses revealed that exposure of the mucosal surface to filipin for 40-80 min in vitro resulted in gross morphological alterations of brush borders in intestinal segments removed from rachitic chicks, but not in preparations from vitamin D-treated birds [60,61]. Moreover Rasmussen et al. [27] have reported that exposure of purified brush border membrane vesicles to filipin resulted in enhanced calcium accumulation in preparations from duodena of rachitic chicks, but not in membrane vesicles isolated from chicks treated with la(OH)D 3, 18 h prior to death. These same authors noted that high levels of filipin (10 t*g/ml) resuited in vesicle destabilization [27], by the criterion of progressive loss of the cation. Vesicle destabilization has also recently been reported to occur in brush border membranes isolated from 1,25(OH)2D3-treated birds, but not in preparations of vitamin D3-treated or deficient chicks [39]. These combined observations, as well as those presented in the following section, indicate that vitamin D status influences one or more fundamental properties of intestinal cell membranes which are likely to involve cholesterol.

311

IIB-2. Phospholipids As noted above, brush border membranes contain far more cholesterol than basal-lateral membranes on a molar basis, an indication that compositional differences reflect functional differences. Phospholipid composition has also been reported to differ between the two plasma membrane fractions [62]. Vitamin D-dependent alterations in the phospholipids of brush border membranes could also lead to localized increases in permeability, perhaps by influencing cholesterol distribution. Green [58] has reviewed the experimental evidence for the preferential association of cholesterol with certain species of phospholipids. The degree of interaction is influenced by the polar head groups of the phospholipids, as well as fatty acyl chain length and degree of unsaturation. Thus, a modification of membrane phospholipids could lead to localized areas of sequestered cholesterol and adjacent regions of greater fluidity. In 1964, Thompson and DeLuca [63] reported a vitamin D-mediated stimulation of phosphate incorporation into the phospholipids of intestinal mucosa. In 1978, Max et al. [38] observed that in chick a portion of the vitamin D-dependent increase in phosphate incorporation occurred in brush border membranes. Moreover, vitamin D treatment altered the ~fattyacid composition of the phosphatidylcholine fraction [38]. Shortly thereafter, O'Doherty [64] reported that intravenous administration of 1,25(OH)2D 3 to vitamin D-deficient rats resulted in enhanced activity of a phospholipid deacylation and reacylation cycle in mucosal cell homogenates. Earlier research had demonstrated that approximately half of the total cellular phospholipase A2, or deacylase enzyme, of rat intestinal epithelium is located in brush borders [65]. Although the subcellular site of the reacyclase activity was not determined, analyses of substrate specificity indicated that arachidonate was most readily transferred to lysophosphatidylcholine, followed by linoleate and oleate [64]. Additional studies by Matsumoto et al. [ 6 6 ] with 1,25(OH)2D3-treated and deficient chicks revealed the seco-steroid increased the de novo synthesis of phosphatidylcholine by stimulation of CDPcholine: sn 1,2-diacylglycerol choline phosphotransferase, by a mechanism independent of

protein synthesis. The time-course of change in brush border phosphatidylcholine: phosphatidylethanolamine ratios after 1,25(OH)2D 3 treatment correlated closely with the time-course of change in calcium uptake into brush border membrane vesicles [66]. Although these workers found no evidence for increased phosphatidylcholine synthesis by methylation of phosphatidylethanolamine, as described by Hirata and Axelrod [67], this pathway might predominate at earlier times (e.g. minutes)after 1,25(OH)2D 3 administration to D-deficient animals, or in normal animals. On the basis of the combined evidence, Matsumoto et al. [66] have postulated that the 1,25(OH)EDa-dependent increase in phosphatidylcholine synthesis acts to provide the preferred substrate for deacylation by phospholipase A E, with subsequent reacylation with more unsaturated fatty acids. The increased content of unsaturated fatty acids results in greater fluidity of the brush border membrane, and thus, greater activity of the calcium transport protein [66]. Further support for the importance of the lipid environment comes from the earlier observation that treatment of brush border membrane vesicles with cis-vaccenic acid in vitro stimulated calcium uptake in preparations from vitamin D-deficient, but not treated chicks, whereas incubations with trans-vaccenic acid decreased uptake in vesicles prepared from 1,25(OH)2Da-treated birds, but not in those prepared from deficient chicks [37] The choice of vaccenic acid may have been fortunate however, as Hill et al. [68] have reported that another cis-fatty acid, linoleate, inhibits the carrier component of a different transport system, i.e. glucose-6-P uptake in liver endoplasmic reticulum, although the glucose-6-phosphatase component is unaffected. Studies have also been conducted on the effect of essential fatty acid restriction on vitamin D-dependent calcium absorption, with conflicting resuits. Lack of dietary unsaturated fatty acids has been reported to inhibit the action of vitamin D on calcium absorption in intestine of rat [69] and chick [42,70] when transport is measured in vitro. However, essential fatty acid deficiency does not inhibit vitamin D-dependent calcium absorption in chicks when transport is measured in vivo [42,70].

312

To date, no reports have been published concerning vitamin D-mediated alterations in brush border phosphatidic acid levels, although this lipid has been found to be a natural calcium inophore in parotid cells [71]. Nor have glycolipids been studied as a function of vitamin D status, although compositional differences in glycolipids exist between brush border and basal-lateral membranes [62]. Finally, under the conditions employed by Max et al. [38], no vitamin D-dependent alterations in brush border phosphatidylinositol were observed. However, rapid turnover of inositol phospholipids has been observed in relation to calcium movement and other membrane-associated phenomena in other cell systems [72,73]. In addition, alkaline phosphatase in microsomes prepared from pig kidney is bound to the membranes by strong interactions with phosphatidylinositol [74]. If a similar anchorage dependence exists for the intestinal brush border hydrolase, it is conceivable that a vitamin D-mediated alteration in associated phosphatidylinositol levels could occur.

llC. Proteins IlC-1. Enzymes A number of brush border enzyme activities are known to be influenced by vitamin D status, lipid environment, or both. a. Guanylate cyclase (EC 4.6.1.2). Guanylate cyclase, which catalyzes the formation of cyclic guanosine 3', 5'-monophosphate (cyclic GMP) is enriched in the brush border fraction of. intestinal mucosa [75-77]. The activity of this enzyme is subject to modulation by lipid environment [78] and is postulated to play a role in intestinal ion transport [79-81]. Although no conclusive evidence exists for vitamin D-dependent calcium or phosphate transport requiring altered guanylate cyclase activity, cyclic G M P levels are increased in duodenal mucosa of chick after 1,25(OH)2D 3 in vivo [82], and may be responsible for the phosphorylation of one or more brush border proteins (see below). b. Alkaline phosphatase (EC 3.1.3.1). Perhaps the most actively studied brush border enzyme is vitamin D-sensitive alkaline phosphatase (cf. Refs. 21,26,33-35,83-91) or calcium-dependent adenosine triphosphatase [32,34,92]. The two catalytic

activities appear to be the property of the same enzyme [34,90,91]. For several years alkaline phosphatase was a promising candidate to be the biochemical entity responsible for vitamin D-stimulated calcium transport. The time-course of elevated alkaline phosphatase activity after vitamin D-administration to D-deficient animals corresponded well with that of vitamin D-dependent calcium transport in early reports [32,33]. However, when similar studies were carried out using 1,25(OH)2D 3, it became apparent that intestinal calcium transport was maximal at 10-14h and the increase in alkaline phosphatase only occurred between 2 4 - 4 8 h [93,94]. These results clearly emphasize the importance of using a form of vitamin D, namely 1,25(OH)2D3, that is not subject to time-consuming metabolism prior to initiating biological effects. Although Bachelet et al. [86] convincingly demonstrated enhanced alkaline phosphatase activity in brush borders after 30 min of perfusion of rat intestine with 1,25(OH)2D 3, an additional dissociation between the hydrolase and calcium transport became apparent. Bikle et al. ([26] cf. Ref. 94) have reported that inhibition of RNA or protein synthesis abolishes the vitamin D-dependent increase in alkaline phosphatase, but does not alter elevated calcium transport. This observation has been confirmed by another laboratory [27]. Since vitamin D is also known to stimulate phosphate uptake in intestine [88, 95-97], a relationship between alkaline phosphatase and uptake of the anion has also been postulated but is subject to the same observation that phosphate uptake, measured in brush border membrane vesicles, is also apparently independent of protein synthesis [88]. Regardless of the actual role alkaline phosphatase plays in the response of intestinal cells to vitamin D, a number of additional intriguing observations have come from monitoring this and other brush border hydrolases. In 1976, Moriuchi and DeLuca [35] found that vitamin D treatment of rachitic chicks resulted in the decrease of a brush border protein having a molecular weight of 200000 and a concomitant increase in a protein with a molecular weight of 230000, that also showed enhanced calcium binding. Moriuchi et al. [84] found the altered molecular weight of alkaline phosphatase to be the result of 1,25(OH)2D ~-

313 TABLE I EFFECT OF VITAMIN D STATUSON THE RELEASEOF BRUSH BORDER HYDROLASESBY PAPAIN AND BROMELAIN Brush borders were isolated from vitamin D-deficient chicks ( - D) or birds treated with 32.5 nmol of vitamin D 3 96, 72, 48 and 24 h prior to death (+ D), and exposed to protease at the final concentrations given in parentheses. Sampleswere taken at various intervals, diluted with ice-cold saline, and soluble and membrane-bound hydrolase activities separated by centrifugation. Values represent the mean of triplicate incubations for three to four independent experiments. Brush border hydrolase

Alkaline phosphatase Leucine aminopeptidase Maltase Sucrase

Papain treatment (0.1 mg/ml)

Bromelain treatment (4 mg/ml)

Time (min)

Time (rain)

20 1 3 20

Hydrolaseactivity released P (percentof total) -D

+D

16.1~1.2 a 28.0~3.0 12.2~0.7 41.0~4.9

14.5±1.3 >0.2 32.1±2.6 <0.01 2 3 . 7 ± 2 . 2 <0.~1 34.9±4.2 0.02

60 10 60 60

Hydrolaseactivity released P (percentof total) -D

+D

70.6±4.7 94.8±2.5 57.9±3.2 60.2~3.1

29.5±4.6 71.0~4.7 36.9~1.7 34.0~2.1

0.~1 <0.~1 <0.001 <0.001

a Deviations represent S.E.

stimulated incorporation of sialic acid into one of the four duodenal isoenzymes. Elevated, calciumdependent sialyltransferase activity in duodenal microsomes was found to precede the appearance of the 230000 duodenal isoenzyme [84]. The existence of alkaline phosphatase isoenzymes in duodenum [98], as well as differential degrees of glycosylation, have been proposed as two possible explanations for the recently reported results of limited proteolysis experiments [99]. These studies were undertaken to determine whether the vitamin D-mediated increase in brush border membrane permeability, regardless of mechanism, results in an altered topography of marker hydrolase activities as judged by accessibility to release by the proteases papain [39] or bromelain [99]. Papaln treatment of brush borders isolated from vitamin D-deficient and replete chicks resulted in the solubilization of only a small portion of the available alkaline phosphatase activity, and failed to reveal any topographical differences mediated by vitamin D status (Ref. 99, Table I). In contrast, treatment of equivalent brush border fractions with bromelain, resulted in lower soluble levels of alkaline phosphatase in preparations from vitamin D-treated than from deficient birds (Table I; Ref. 99). Analyses for recovery of activity after proteolysis indicated no loss of alkaline phosphatase activity after papain treatment,

but a selective, bromelain-mediated inactivation of the alkaline phosphatase associated with brush borders of vitamin D-treated chicks [99]. Alternative explanations for the observed inactivation were loss of an activator, such as calcium-binding protein [100], or modified insertion of the hydrolase in the lipid bilayer [99]. An attempt to investigate the effects of bilayer perturbation was made by treating isolated brush borders with filipin [99]. The polyene antibiotic had very little effect on either the specific activity of membrane-bound alkaline phosphatase in preparations from vitamin D-deficient [99] or treated birds (Nemere, unpublished data), or on cleavage and release by bromelain from brush borders of rachitic chicks [99]. c. Leucine aminopeptidase (EC 3.4.1.3). Another marker hydrolase that was monitored in the experiments on limited proteolysis (see above) was leucine aminopeptidase. Vitamin D-treatment of chicks was found to produce a small, but significant increase in brush border leucine aminopeptidase specific activity. As indicated in Table I, papain treatment of isolated brush borders resuited in small but significant increases in soluble leucine aminopeptidase levels in preparations from vitamin D-treated birds, relative to rachitic controis [39] whereas bromelain treatment gave the opposite result [99]. Neither protease diminished leucine aminopeptidase recovery from 100%

314 [39,99], nor did filipin treatment in vitro influence solubilization or specific activity of membrane-associated leucine aminopeptidase [99]. d. Maltase (EC 3.2.1.20). Earlier studies on the effect of vitamin D status on brush border hydrolases failed to detect a change in maltase specific activity [83]. Using purer preparations, Nemere and Norman [39,99] recently reported a statistically significant decrease in maltase specific activity in brush borders, as well as in whole homogenates, of duodenal epithelium from vitamin D-treated chicks. Vitamin D status was also found to influence the extent of proteolytic release of this disaccharidase from isolated brush borders. Maltase was more efficiently released by papain and less efficiently by bromelain from brush borders of vitamin D-treated chicks, relative to paired preparations from rachitic chicks (Table I). Although filipin pretreatment of brush borders in vitro once again had little effect on disaccharidase release by bromelain, the polyene antibiotic mediated a 30% decrease in the specific activity of membrane-bound mahase [99], comparable to the decrease mediated by vitamin D in vivo. e. Sucrase (EC 3.2.1.26). Max et al. [38] reported a large, vitamin D-mediated decrease in sucrase activity of brush border membrane vesicles prepared from chick duodenum. This observation has been confirmed by other workers [39,42,99]. Despite the drastic decrease in sucrase, and the existence of hydrolase-related glucose transport in intestine [101], vitamin D has been found to enhance glucose uptake in organ cultures of embryonic chick duodenum [87], as well as jejunum and ileum of chick [102]. In light of the finding that 1,25(OH)2D 3 stimulates insulin release from the pancreas [103], and that circulating insulin levels modulate certain intestinal functions such as sucrase activity [104] and glycosylation of microvillar membrane proteins [105], it remains to be determined which hormone(s) is(are) directly responsible for effects reported as 'vitamin D-mediated'. Among the observations on vitamin D-mediated effects on sucrase, is the lower extent of solubilization of the disaccharidase by either papain or bromelain treatment of brush borders isolated from vitamin D-treated birds, relative to rachitic controls (Refs. 39,99; Table I). Moreover, treatment of

brush border preparations with filipin in vitro also inhibited membrane-bound sucrase specific activity I99]. The differential effect of filipin on the extrinsic [ 106,107] membrane proteins, maltase and sucrase, but not leucine aminopeptidase or alkaline phosphatase, strongly suggests the existence of cholesterol-rich microdomains (cf. Ref. 58), and supports the importance of lipid environment in the modulation of protein functions.

HC-2. Calcium-binding proteins Within the course of the limited proteolysis experiments described above, the effect of papain treatment of brush border membrane vesicles on calcium uptake was studied [39]. Rather than diminishing calcium uptake, limited proteolysis enhanced accumulation of the cation, regardless of the vitamin D status of the chicks from which the vesicles were prepared [39]. This effect of proteolysis can most likely be attributed to a generalized increase in fluidity resulting from removal of membrane proteins (cf. Ref. 58,108), which in turn might allow a conformational change in calcium transport components. This phenomenon has been observed for another carrier-mediated transport system: Berteloot et al. [109] have found that exposure of intestinal brush border membrane vesicles to papain results in a 2-fold increase in glucose uptake, relative to native vesicles. The failure of papain treatment of isolated brush borders to inactivate a vitamin D-induced calcium transport component could be attributed to the substrate specificity of the enzyme, a n d / o r intrinsic localization of a protein transport component. A number of workers have reported the isolation of calcium-binding proteins from brush borders of rat intestine, and have suggested that such proteins, distinct from cytoplasmic calcium-binding protein, are components of the calcium transport complex [31,35,110-112]. Other workers have simply recorded the observation that brush borders [60] or membrane vesicles [113,114]; prepared from vitamin D-treated animals bound more calcium than equivalent fractions from vitamin D-deficient animals. One of the most extensive investigations of vitamin D-dependent calcium-binding proteins of intestinal brush border membranes has been con-

315

ducted by Kowarski and Schachter [112]. These workers initially isolated a vitamin D-dependent calcium-binding complex of high molecular weight containing maltase, calcium-dependent ATPase, and p-nitrophenylphosphatase activities [ 112]. The calcium-binding activity has recently been separated from the enzyme activities [111]. Initial characterization of the calcium-binding protein (IMCal) has revealed a M r of 200000, which can be dissociated to yield monomers of M r 20500 [111 ]. The calcium-binding activity was previously shown to correlate positively with known features of intestinal calcium transport mechanism, i.e., distribution in the small intestine, effects of vitamin D, dietary calcium, and rat age [31]. The authors also reported that the vitamin D-dependent increase in calcium-binding complex required protein synthesis. This observation has not been reconciled with the findings of Bikle et al. [26] and Rasmussen et al. [27]. A further problem arises upon consideration of affinity for calcium. In 1975, calcium-binding complex was reported to have a pKca approx. 4.7 or an apparent K D of 2 . 1 0 -5 M, and was postulated to act by transferring calcium to the higher-affinity calcium-binding protein of the cytoplasm [31 ]. The apparent disassociation constant for ImCal has been reported to be 3.7.10 -7 M, as compared to 2.25. 10 - 6 M for soluble calcium-binding protein [111]. These relative affinities suggest the need for a modulating factor or a conformational change that would promote the transfer of calcium from ImCal to calcium-binding protein, in order for such a translocation mechanism to function.

HC-3. Other proteins Wilson and Lawson [115] have reported an increase in a brush border membrane protein with a molecular weight of 84000 that is believed to be a subunit of alkaline phosphatase [116]. Other workers have observed a vitamin D-mediated increase in levels and radioactive labelling of proteins with molecular weights ranging from 76000 to 90000 [38,42,117]. Evidence also exists to indicate that vitamin D mediates an increase in proteins with molecular weights of 133000 and 233000, as well as a decrease in proteins with molecular weights of 83000 and 111000 [118]. In

addition, vitamin D has been found to enhance phosphorylation of protein(s) within the 80-kDa region [118,119]. Two other proteins with molecular weights of 17000 and 42000 have been shown to undergo vitamin D-dependent alterations as judged by decreased labelling with iodination reagents [118]. Within this same series of experiments, the 17-kDa protein was localized on the lumenal surface of brush borders, and therefore is not calmodulin, whereas the 42-kDa protein was identified as core material actin (see below).

HC-4. Cytoskeletal proteins The electron dense core material of intestinal microvilli is known to consist predominantly of actin polymers, or filaments, that have uniform polarity with respect to the microvillus tip [121]. Wilson and Lawson [115] have found that 1,25(OH)2D 3 administration to chicks in vivo stimulates the incorporation of [3H]leucine into actin during in vitro incubations of jejunal slices from chicks. The vitamin D-dependent increase in actin labelling has not been demonstrated to give rise to increased levels of the protein as judged by densitometric scans of brush border proteins separated by gel electrophoresis [42]. Lack of synthesis in the presence of accentuated labelling could be a result of rapid turnover of actin monomers ( M r 42,000) or polymerization-depolymerization cycles, that perhaps are directly controlled by 1,25(OH)2 D 3 [ 122]. Support for the rapid turnover of actin monomers comes from the finding that another core protein, villin ( M r 95000), is a calcium-sensitive ( K d • 2.5 • 10 - 6 M ) regulator of actin polymerization [123,124]. In the presence of low calcium ( < 10 7 M ) villin promotes actin polymerization, whereas calcium levels exceeding 10 - 7 M promote villin-regulated depolymerization of actin [ 123,124]. These data have an obvious implication for vitamin D-enhanced uptake of calcium by mucosal cells. The sensitivity of villin-actin complexes to calcium suggests the necessity of sequestering calcium during the uptake process, perhaps by another core protein such as calmodulin (cf. Ref. 123) which is abundant in rat intestine, but unaffected by vitamin D status with respect to localization along the length of the intestine [125]. A small amount of vitamin D-dependent calcium-

316

binding protein has also been identified in microvilli [52]. An alternative means of sequestering calcium could be in membrane delimited organelles (see below). Although the exact function of the contractile protein network in intestinal brush borders remains obscure, it has been proposed [121] that the network is responsible for microvillar motility [126] and the transport of endocytic vesicles [124]. The actin filaments penetrate the terminal web [121], which has been shown to also contain actin microfilaments, as well as filamin, tropomyosin, a-actinin (cf. Ref. 124) and vitamin D-dependent calcium-binding protein [52,127] as judged by immunocytochemical techniques. Addition of ATP and calcium to isolated brush borders results in a contraction of the terminal web region, without concomitant microvillar movement or shortening (cf. Ref. 121). Thus, the terminal web region of the intestinal cytoskeleton may also be involved in transport functions. Fuchs and Peterlik [128] have attempted to determine the role of cytoskeletal structures in vitamin D-induced transepithelial phosphate and calcium transport in everted sacs of chick jejunum. Treatment of the mucosal surface with the microfilament-disrupting agent, cytochalasin B, did not alter phosphate influx into cells, but did abolish vitamin D-enhanced transfer of phosphate to the serosal side [128]. Similar concentrations of cytochalasin B decreased vitamin D-enhanced calcium uptake and translocation, but did not completely suppress either absorption component to levels observed for preparations from rachitic chicks [128]. Disruption of microtubules by colchicine had no significant effect on either phosphate or calcium transport [128]. The apparent lack of effect of cytoskeletal inhibitors on vitamin D-stimulated calcium translocation is difficult to interpret in light of the findings that certain agents in these categories actually promote release of intracellular vesicles [129,130], and that microtubules contribute to the maintenance of the polarity of intestinal epithelial cells with respect to membrane glycoprotein distribution [131]. Although a considerable amount of attention has been focused upon the intestinal brush border as the principal regulatory site for vitamin D-

stimulated calcium transport, a recent report by Shultz et al. [132] suggests the existence of additional control points. These workers studied calcium transport in vitamin D-deficient chicks treated with vehicle, 1,25(OH)zD3, or 1,25(OH)2D 3 and cortisol, using the ligated loop technique in situ, as well as accumulation of the cation into brush border membrane vesicles isolated from similarly treated birds. The results indicated that cortisol diminished 1,25(OH)2D3-dependent calcium transport in vivo, but failed to significantly alter accumulation of the cation into brush border membrane vesicles [132]. Thus, it is essential to consider the potential contribution of other cellular components to vitamin D-dependent calcium absorption.

I11. Intracellular organelles I l i A . Mitochondria

The cytotoxic effects o f high intracellular calcium concentrations has contributed to more than one proposal that intracellular organelles sequester the cation, particularly during periods of influx. Mitochondria have for years been a favorite candidate for the proposed role, as they are known to accumulate excessive amounts of calcium in vitro, apparently by way of a chymotrypsin-sensitire protein [133]. Some evidence suggests 1,25(OH)zD 3 treatment of rachitic chicks stimulates the production of a mitochondrial outermembrane protein in advance of maximum calcium transport [134]. However, other evidence suggests this protein has a microsomal origin [116]. Electron microscopy has been used to support the proposed role of mitochondria in vitamin D-mediated calcium transport. One study found that calcium deposits, visualized by osmium tetroxide staining, were limited to the intestinal microvillus region in vitamin D-deficient rats, but were predominantly observed in mitochondria of vitamin D3-treated and normal rats [135]. Other correlations between mitochondria, intracellular calcium levels, and vitamin D status have been reviewed elsewhere [94]. Despite such correlations, several studies argue against mitochondria as mediators of vitamin Dstimulated calcium translocation. Bikle et al. [94]

317

have observed the absence of increased calcium accumulation by mitochondria isolated after 1,25(OH)2D 3 treatment in vivo, relative to preparations from rachitic chicks, and the inability of the organelles to accumulate calcium in vitro at levels believed to exist in the cytosol. Moreover, localization of intracellular calcium by electron probe microanalysis (EPMA) has failed to detect significant a c c u m u l a t i o n of calcium in mitochondria of various tissues [48,53,136,137]. Denton et al. [138] have reported a biphasic effect of calcium on mitochondrial oxidative metabolism, and suggest that the calcium uptake and release system in these organelles is more likely to be involved in the control of intramitochondrial rather than cytoplasmic levels of the cation. IIIB. Vesicles In 1975, an extensive study of intestinal calcium absorption using EPMA was conducted by Warner and Coleman [48]. These workers analyzed duodenal, jejunal and ileal segments of rat and chick intestine, as well as comparing duodenal segments from normal, vitamin D-deficient and treated animals [48]. It was reported that under conditions of maximal active transport and minimal passive transport, intracellular calcium was found in discrete localizations in absorptive cells primarily along the lateral membranes. A decreased number of absorptive cell localizations were observed in tissue from vitamin D-deficient rats, and an increased number of localizations in vitamin D-replete rats [48]. Calcium concentrations were not normally encountered within the microvillar border or mitochondria, regardless of vitamin D status [48]. Moreover, Warner and Coleman [48] performed a number of control studies that convincingly demonstrated that preparative artifacts, such as fixation with osmium tetroxide and oxalate, promoted a diffuse calcium distribution intracellularly and even extracellularly. Further support for localized calcium deposits in intestinal cells comes from the observation that vitamin D-treatment also increases vesicular calcium in chondrocytes [139]. The transit path of the calcium localizations in the intestinal cells was reported to be internalization near the junctional complex just below the terminal web and move-

ment along the lateral border, or translocation to a supranuclear position and then to the lateral-basal region of the absorptive cell [48]. On the basis of these observations, other workers have either identified the sequestering organelles as Golgi vesicles or lysosomes (see below). The different identifications may be, but need not be, mutually exclusive, as the Golgi apparatus is known to "package" lysosomal enzymes [140,141] and such primary lysosomes migrate with other Golgi vesicles during density centrifugation. Also, lysosomes have been found to fuse with other Golgi vesicles in a calcium-dependent manner [142]. In any event, the studies discussed below are indirectly supported by the finding that hormones such as glucagon and insulin increase and decrease calcium uptake by "microsomes" isolated from liver [143]. Of more direct interest are the reports by Feher and Wasserman [144-146] of a membrane-bound fraction of vitamin D-induced calcium-binding protein. Sorbitol density centrifugation failed to conclusively identify which fraction the membrane-bound calcium-binding protein is associated with, but eliminated the basal-lateral, mitochondrial, and brush border fractions [145]. IIIB-1. Golgi vesicles In 1977, Freedman et al. [147] fractionated intestinal cell membranes by density gradient centrifugation, and obtained Golgi, basal-lateral, and microvillar membranes enriched in galactosyltransferase, (Na ÷ +K÷)-ATPase, and sucrase activities, respectively. Uptake studies indicated that the Golgi vesicle fraction accumulated by far the highest calcium levels of the membranes tested [147]. Kinetic studies with Golgi vesicle fractions isolated from vitamin D-deficient rats revealed a 94% decrease in the initial rate and an 81% decrease in equilibrium levels of calcium uptake, relative to equivalent fractions from normal rats. Unfortunately, the authors have yet to present data on uptake capacity at calcium concentrations believed to exist in cytosol (10-8-10 -7 M) [147149], nor have they conclusively determined the cause for the vitamin D-dependent difference in equilibrium value. However, electron micrographs of the Golgi membrane fractions did not indicate a vitamin D-dependent difference in vesicle size [147].

318

Subsequent studies from the same laboratory demonstrated.a biphasic recovery of calcium uptake in Golgi vesicles isolated from vitamin D-deficient rats that were given a single intrajugular injection of 1,25(OH)zD 3. A rapid initial phase was evident 15-30 min after administration of the steroid hormone, followed by a decline to prestimulated levels at 8 h and a late recovery phase between 48 and 96 h after 1,25(OH)2D 3 [150]. The second recovery phase correlates well with the time period required for a renewal of the intestinal cell population (151, cf. Ref. 152). By comparison, calcium uptake recovery in everted gut sacs became noticeable well after the initial recovery observed in the Golgi fraction, but remained maximally elevated between 6 and 120 h after 1,25(OH)2D 3 in vivo [150]. The lack of temporal correlation between recovery in isolated Golgi vesicles and intestinal segments led the authors to suggest that an additional factor is necessary for 1,25(OH)2 D3-mediated calcium uptake [ 150]. MacLaughlin et al. [150] have postulated that the early recovery phase of Golgi vesicles in 1,25(OH)2D 3dependent calcium uptake may be mediated by cAMP, since adenylate cyclase is present in the Golgi fraction [153], and 1,25(OH)2D 3 has been reported to stimulate a biphasic increase in cAMP in organ cultures of embryonic chick intestine, with the first phase apparent by 30 rain after exposure to steroid [154]. Further investigation of uptake parameters in isolated Golgi vesicles led to the conclusion that most of the accumulated calcium was bound to internal components ( K a = 3 . 8 . 1 0 - 6 M), as the extent of uptake was found to exceed available intravesicular space [148]. This conclusion suggests that the vitamin D-dependent differences in calcium accumulation [147] reflect an alteration in vesicular constituents. It is conceivable that the vitamin D-mediated increase in calcium uptake by Golgi membranes occurs through the packaging of more acidic vesicular components. Indeed, Freedman et al. [148] have observed that a proton gradient stimulates the rate of calcium uptake, when the intravesicular pH is more acidic than that of the incubation medium. The findings suggest that the rate of in situ calcium accumulation would be optimal if the intravesicular environment is at pH 4-5. The authors suggest that the vitamin

D-mediated increase in calcium binding sites in Golgi vesicles could be attributable to the packaging of vitamin D-dependent calcium-binding protein [148]. Recently, Weiser et al. [155] have observed that accumulation of calcium is greatest in Golgi vesicles isolated from duodenum, and decreases in equivalent preparations from jejunum and ileum. These workers have also reported that uptake is not dependent on the presence of ATP, but does require protein synthesis [155]. The Golgi apparatus might also contribute to other vitamin D-related phenomena that are not directly connected with calcium transport. As an organelle that is enriched in glycosyltransferases, Golgi vesicles are probably the source of the vitamin D-enhanced sialyltransferase that increases sialic acid incorporation into alkaline phosphatase [84]. The observation of vitamin Denhanced glucose Uptake [102], might also be attributable to vesicular flow in a manner analogous to that of the action of insulin on adipose tissue. Cushman and Wardzala [156] have reported that insulin stimulates glucose uptake by promoting the translocation of the transport system from an intracellular microsomal membrane pool to the cell surface. Moreover, by analogy to insulin action in adipocytes, the Golgi apparatus of intestinal cells might also be the source of the vitamin D-dependent calcium transport complex of brush border membranes.

IIIB-2. Lysosomes The direct evidence for the lysosomal mediation of vitamin D effects is not as extensive as the indirect evidence. However, it has been demonstrated that treatment of isolated intestinal cells from normal rats with 65 pM 1,25(OH)2D 3, results in an early (15-30 min) and significant increase in the release of lysosomal hydrolases and alkaline phosphatase, relative to vehicle controls, and without loss of cell viability [89,157]. The acute release of the hydrolases was temporally correlated with an equally rapid uptake of isotopically labelled calcium followed by an apparent extrusion of the radionuclide [157]. The observation that alkaline phosphatase was released with the lysosomal hydrolases studied (i.e., acid phosphatase, cathepsin B, N-acetyl-/3-D-glu-

319 cosaminidase) gained support by the biochemical evidence that the alkaline hydrolase is enriched in the same subcellular fraction as the acid hydrolases [89], as well as by the electron microscopic studies of Hugon and Borgers [158]. It has also been postulated that 1,25(OH)2D3-induced vesicular flow contributes to the early increase in brush border alkaline phosphatase reported by Bachelet et al. [86]. Moreover, the finding that rat intestinal epithelial cells have at least two functionally discrete subpopulations of lysosomes [89], coupled with observations on vesicular flow [131,159], suggests a lysosomal origin for the acid phosphatase activity [160] associated with isolated basal lateral membranes [40]. Exocytosis of lysosomal enzymes could also very well be responsible for the 1,25(OH)2Da-mediated decrease in fibroblast adhesion [161], perhaps through enzymatic alterations of an attachment protein such as fibronectin [162]. Enzymatic alteration of intestinal crypt cell fibronectin [163] could also account for vitamin D-enhanced cell migration up the villus (cf. Ref. 152). Davis et al. [53,136] have reported that the discrete calcium localization sites of intestinal absorptive cells are lysosomes. In a study of normal chick duodenum using EPMA, microincineration, EGTA chelation, and histochemical staining techniques for electron microscopy, these workers observed dense calcium deposits in vesicles that stained positively for acid phosphatase [53,136]. In a subsequent study, Davis and Jones [136] found that supranuclear, calcium-sequestering lysosomes of vitamin D-replete animals were larger and more numerous than those of rachitic animals. The calcium-containing lysosomes of vitamin D-replete animals had the appearance of multi-vesicular bodies filled with pinocytic vesicles, and were close to, but not in, the terminal web [136]. By comparison, lysosomes of rachitic animals were smaller, did not have a compound appearance, and were generally further from the terminal web region [136]. Unlike Warner and Coleman [48], Davis et al. [53,136] observed calcium deposits in mitochondria and in microvilli, with heavier deposits in microvilli of rachitic chicks. Although Davis and co-workers did not discuss the relative merits of various fixation and staining procedures or the possible occurrence of redistribution

artifacts, similar results were reported for three different procedures [53]. A lysosomal origin of calcium released during bursting activity in snail neurons [164] has also been reported. The combined data from rat, chick, and snail suggest that sequestering calcium within lysosomal vesicles is a cellular function that is highly conserved in an evolutionary sense. IV. Basal lateral membranes

The difficulty of isolating pure basal lateral membrane preparations is reflected in the paucity of reports concerning the effects of vitamin D on this subcellular fraction. It is known, however, that basal lateral membrane vesicles prepared from normal rats, exhibit greater calcium uptake than equivalent preparations from rachitic animals [147] presumably through the activity of a high affinity Ca-ATPase present in such fractions [165-167]. Also, since adenylate cyclase is associated with basal lateral mambranes [75,153], this enzyme might contribute to 1,25(OH)2D3-enhanced levels of cAMP in organ cultures of embryonic chick intestine [ 154]. Mircheff et al. [168], and more recently Ghijsen and Van Os [169] have reported that a Ca-ATPase enriched in basal lateral membranes of rat duodenum, exhibits a 1,25(OH)2D3-dependent increase in activity. Potential mechanisms for activation of this enzyme are suggested by work on the Ca-ATPase of erythrocyte membranes [170]. In the erythrocyte model system, Ca-ATPase is known to be stimulated by calmodulin, through a shift from a low Ca affinity form ( K D = 15 /~M) to a high affinity form ( K D = 0 . 4 /~M [170]). Calmodulin has also been reported to enhance the affinity of the Ca-ATPase activity in basal lateral membrane fraction of rat small intestine, as well as the maximal transport rate of the cation [166]. However, since vitamin D does not mediate an increase in calmodulin levels [125] in intestinal cells, it is interesting to note that in the erythrocyte system, the shift to a high affinity form occurs in the presence of acidic phospholipids [170], some of which are known to bind calcium [171]. The shift in affinity can also be induced by limited proteolysis (cf. Ref. 170).

32() V. M e c h a n i s m s

INTESTINAL

Figs. 1-3. i l l u s t r a t e e l e m e n t s of s o m e p o t e n t i a l m e c h a n i s m s for v i t a m i n D - m e d i a t e d i n t e s t i n a l t r a n s p o r t o f c a l c i u m . A l t h o u g h the m e c h a n i s m s are d i v i d e d i n t o t w o b r o a d c a t e g o r i e s , it is likely t h a t e l e m e n t s f r o m e a c h c a t e g o r y c o n t r i b u t e to the actual c e l l u l a r e v e n t s of c a l c i u m t r a n s p o r t . I n d e e d , a p r o p o r t i o n of the d a t a p r e s e n t e d in earlier sections of this r e v i e w c a n be i n t e r p r e t e d in f a v o r of m o r e t h a n o n e m e c h a n i s m . F o r this reason, an a t t e m p t has b e e n m a d e to i d e n t i f y s i g n i f i c a n t s t r e n g t h s a n d deficiencies.

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: UNOCCUPIED 1,25(OH)2D3 RECEPTOR = OCCUPIED 1,25{OH)20 ~ RECEPTOR

Fig. I. Protein carrier mechanism of intestinal calcium transport at the level of the epthelial cell, comparing some known aspects of the effect of filipin in vitro (left panel) or 1,25(OH)2D 3 in vivo (right panel) with calcium transport in the vitamin D-deficient animal (central panel). In this model, the vitamin D-deficient state is characterized by a low level of calcium transport from the lumen, across the brush border, and into the epithelial cell. Unoccupied receptors for 1,25(OH)2D 3 are present in the cytoplasm and nucleus, and the basal lateral membrane contains Ca-ATPase for extrusion of the cation, in exchange for sodium. In the vitamin D- or 1,25(OH)2D3-replete state, the steroid hormone occupies specific receptors yielding complexes that induce the synthesis of calcium-binding protein (CaBP), which in turn binds and transports excess cytoplasmic calcium towards the vascular system. In addition, the right panel illustrates the presence of calcium-binding protein in the nucleus, terminal web, and microvilli, vitamin Dmediated lengthening of the microvilli, and enhanced movement of calcium across the brush border. Filipin similarly increases calcium transport across the brush border, presumably by binding to cholesterol to accentuate membrane fluidity and thus effect greater activity of a transport protein. Unlike the vitamin D-replete state, filipin treatment of intestinal segments removed from vitamin D-deficient animals does not alter receptor occupancy, nor induce the synthesis of calcium-binding protein.

BORDER

= C a B P + Ca 2+

q = CHOLESTEROL

A. P ' T A S E = A L K A L I N E P H O S P H A T A S E

Fig. 2. Detailed aspects of the protein carrier mechanism of calcium transport at the level of a single intestinal epithelial cell brush border microvillus. In the vitamin D-deficient state (left panel), calcium channels or gates exist in a relatively inactive conformation. In addition, alkaline phosphatase levels are low and sucrase levels high in comparison to the vitamin D-replete state (right panel). Although vitamin D status does not greatly alter leucine aminopeptidase activity, the topographical modification is depicted as a consequence of experimental observations [99] (see text). Vitamin D-dependent calcium uptake results from a postulated conformational change(s) in a calcium gate or channel, possibly arising from greater membrane fluidity or the presence of the large, membrane-associated calcium-binding complex, lmCal [111].

VA. Protein carriers Mechanisms involving calcium-specific carriers t h a t are l o c a l i z e d in b r u s h b o r d e r s h a v e a l o n g a n d d u r a b l e s t a n d i n g . T h e c a r r i e r c o n c e p t is b a s e d u p o n o b s e r v a t i o n s that v i t a m i n D - d e p e n d e n t c a l c i u m t r a n s p o r t is a s a t u r a b l e process, i.e., a f i n i t e n u m b e r of h i g h a f f i n i t y sites a r e i n v o l v e d , a n d i n c r e a s e d a b s o r p t i o n o c c u r s w i t h o u t a n alterat i o n in the a f f i n i t y ( K m) of s u c h sites, b u t r a t h e r b y an e n h a n c e d r a t e o f t r a n s l o c a t i o n (Vmax). A v i t a m i n D - m e d i a t e d i n c r e a s e in the n u m b e r of a c t i v e c a r r i e r entities is b e l i e v e d to b r i n g a b o u t the a l t e r e d t r a n s l o c a t i o n rate. R e p o r t s t h a t n e i t h e r a c t i n o m y c i n D n o r cycloheximide inhibit vitamin D-mediated calcium t r a n s p o r t in vivo, suggest that p a r t o r all o f the a b s o r p t i o n steps are n o t d e p e n d e n t u p o n R N A or p r o t e i n s y n t h e s i s [26,27]. S u c h f i n d i a g s s h o u l d be i n t e r p r e t e d w i t h c a u t i o n since it is k n o w n t h a t b o t h a n t i b i o t i c s affect o t h e r c e l l u l a r c o m p o n e n t s .

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VESICLE

LYSOSOME LYBOSOME BODY

Fig. 3. Vesicular carrier mechanisms of calcium transport. In the left panel, calcium movement across the brush border is postulated to occur through the action of a plasmalemma transport protein (see Fig. 2). The internalized cation is transferred to calcium-binding protein, which in turn delivers the calcium to Golgi vesicles. Transport is completed by the fusion of Golgi vesicles with the basal lateral membrane and exocytosis of calcium. In the right panel, the major entry route of calcium is illustrated as occurring through pinocytic vesicles. Two potential fates of these vesicles are shown: direct fusion with the basal lateral membrane and exocytosis of the cation, or fusion with primary lysosomes (packaged by the Golgi apparatus) to yield secondary lysosomes, including multivesicular bodies. The calcium-bearing lysosomes, in turn, fuse with the basal lateral membrane to complete the transport cycle.

For example, actinomycin D stimulates the activity of intestinal brush border alkaline phosphatase (cf. Refs. 26,172) and cycloheximide has been found to alter the properties of membrane bound vesicles [173,174]. However, as discussed above, studies with the polyene antibiotic filipin suggest that calcium uptake, translocation, and efflux components are present in the intestinal cells of rachitic chicks and that transport is initiated by perturbation of the brush border membrane. More recently, vitamin D-mediated alterations in brush border membrane phospholipid composition have been postulated to account for increases in active cartier entities [88]. The lack of a vitamin D-mediated alteration in membrane fluidity as judged by ESR spectroscopy [42] need not disqualify such a mechanism. Enrichment of the saturated fatty acid content in plasma membranes of cultured cells fails to reveal altered fluidity by ESR spectroscopy, yet suppresses the activity of intrinsic membrane proteins such as adenylate cyclase [175].

As discussed earlier in this review, a number of high-affinity calcium-binding proteins in intestinal brush borders have been reported. In spite of the attention directed to such proteins, no one has given definitive proof of having isolated the carrier. Convincing evidence for such a claim would be to functionally reconstitute the protein components of the carrier into liposomes, and demonstrate uptake capacity. In the classical model of transport, the newly acquired calcium is transferred from the membrane-bound carrier to a soluble calcium-binding protein, e.g. vitamin D-dependent calcium-binding protein, in order to prevent the saturation of every available intracellular binding site. A consideration of the calcium-sensitive microfilament proteins in brush borders is pertinent at this point. If the membrane-bound carrier is localized on the microvilli, a calcium-buffeting mechanism must exist to prevent the disintegration of the brush border. Preliminary experiments suggest that microvillar core proteins isolated from vitamin D-treated animals are indeed more stable in the presence of 10-6-10 -3 M CaC12 than preparations from vitamin D-deficient animals [42]. The stabilizing factor remains to be identified though, since no protein component corresponding to vitamin D-induced calcium-binding protein has been found when brush border proteins are separated by SDS-gel electrophoresis [42]. Alternatively, if the membrane-bound carrier is localized at the base of adjacent microvilli, calcium could more readily be transferred to vitamin D-dependent calcium-binding protein in the terminal web [52,127]. Although immunoreactive calciumbinding protein is not detectable at the onset of vitamin D-stimulated absorption, it has not been determined whether the available antisera recognize the protein in the absence of bound calcium, since cation binding is accompanied by a conformational change [176] that might affect available antigenic sites. Moreover, in the absence of calcium, calcium-binding protein is susceptible to proteolytic cleavage by trypsin-like enzymes [176]. The intriguing possibility exists that one or more proteolytic fragments represent an active form, and vitamin D stimulates the synthesis of an antigenically-distinct storage form of M r 28000 in chick.

322 Regardless of the identity of the proposed cytoplasmic carrier protein, there is evidence for a vitamin D-mediated increase in cytoplasmic calcium. Using EPMA to analyze the distribution of calcium in intestinal absorptive cells, Warner and Coleman [48] found that although levels of the cation in the apical cytoplasm were near the limit of resolution for the technique, samples from normal and vitamin D3-treated rats had 2-4-fold more calcium than those from vitamin D-deficient animals. However, if vitamin D-stimulated calcium transport does occur through such a cytoplasmic route, additional questions concerning translocation mechanisms arise. For example, movement by diffusion is rapid in model systems, but may not be optimal for a carrier protein that is miniscule in relation to the size of the cell, and must largely bypass the organelles that occupy much of the intracellular space. Movement of the carrier by cytoplasmic streaming through channels might obviate such concerns, but remains to be demonstrated for the calcium carrier protein. Efflux of the ion across the basal lateral membrane has been postulated to be effected by a vitamin D-stimulated Ca-ATPase activity, present at levels that are sufficient to remove excess cytoplasmic calcium [168,169]. The mechanism of calcium transfer from carrier protein to 'pump' has not been proven. However, one intriguing possibility is that the carrier protein loosely binds to the Ca-ATPase, and in doing so, the carrier undergoes a conformational change to effect transfer of the cation (R.J.P. Williams, personal communication), with subsequent exchange across the membrane for sodium [167]. VB. Vesicular carriers

Before undertaking an evaluation of subcellular vesicles, e.g. Golgi, lysosomal, pinocytic, it is necessary to consider what is known about membrane turnover or vesicular flow in cells, particularly those of the intestinal epithelium. Several studies of intestinal cells have demonstrated that renewal of the plasma membrane, as well as the incorporation of intrinsic glycoproteins, occurs through fusion of Golgi-derived vesicles with the plasma membrane in an apparently continuous process [131,159]. Black et al. [177] have reported the

release of microvillus membrane vesicles, bearing alkaline phosphatase and maltase activities~ from embryonic duodenal tissue of chick, both in organ culture and in situ. Endocytosis, especially the formation of pinocytic vesicles (65 nm diameter), is a form of uptake that occurs in most, if not all, cell types, including kidney [178] and intestine ([53,89] cf. Refs. 131,157) and is closely coupled to exocytosis [53,89,157,178-180] as a mechanism for recycling membranes. Thus, despite the static appearance of intestinal cell membranes in electron micrographs, biochemical evidence indicates a constant dynamic flow of membrane components, and any model of vesicular calcium transport must take this into account. In proposing Golgi vesicles as a component of transport in vitamin D-mediated calcium absorption, MacLaughlin et al. [150] did not elaborate upon a means of transferring extracellular calcium to the vesicles. Warner and Coleman [48] postulated that entry might occur through the junctional complex, as EPMA revealed calcium localizations in this area. Golgi vesicles situated near the junctional complex could take up excess intracellular calcium, move to the basal lateral membrane, and release the sequestered calcium by fusing with the plasma membrane. The mechanism has the advantage that once the cation is sequestered, other calcium-sensitive structures within the cell are protected during transport. However, calcium movement through the junctional complex results in functional uncoupling of cell-cell communication [181 ]. Whether or not such uncoupling is desirable, its occurrence should be detectable by electron microscopic examination of freeze-fracture replicas of gap junctions [181]. Another point that requires resolution, is the efficiency of calcium uptake by Golgi vesicles in vivo during vitamin D-stimulated absorption. Freedman et al. [148] have reported that uptake by isolated vesicles apparently occurs by a carrier mechanism, with K D ----3.7. 10 6 M. It remains to be demonstrated that the affinity of Golgi vesicles for calcium is sufficient to prevent indiscriminate binding of the cation to cytoskeletal elements. Alternatively, soluble transport proteins could deliver calcium from the brush border carrier complex (see above) to Golgi vesicles. An efficient means of calcium uptake could

323 occur by pinocytosis [53]. Since a number of membrane proteins have been found to be internalized during the formation of vesicles [179,180,182], it is conceivable that a brush border membrane protein with a preferential affinity for calcium, upon binding sufficient cation, would be internalized in a manner analogous to hormone receptors (cf. Ref. 183). Indirect support for such a mechanism comes from the finding that treatment of embryonic chick duodenum in tissue culture with exogenous, vitamin D-dependent calcium-binding protein stimulates calcium uptake, as well as mucosal-to-serosal transport [184]. The calcium ionophore A23187 has also been found to stimulate vesicle formation [185-187]. Additional support comes from the observation that endocytosis is suppressed under conditions of cholesterol depletion [188] and strongly affected by the phospholipid composition of the plasma membrane, with respect to saturation of fatty acid side chains and type of polar head groups [ 189]. Uptake by an endocytic process would obviate problems of calcium binding to cytoskeletal components and other organelles, and need not contradict the observations of Warner and Coleman [48]. Since pinocytic vesicles have a diameter of 65 nm, a calcium-bearing vesicle would probably not appear as a dense localization. Calcium localizations would become apparent when the pinocytic vesicles concentrated in one region of the cell a n d / o r fused with lysosomes [53]. The two potential fates of the calcium-bearing vesicles, direct fusion with the basal lateral plasma membrane or an intermediate fusion with lysosomes, each have some experimental support. The first alternative, direct delivery of calcium, might be feasible on the basis of studies with model systems. Zimmerberg et al. [190] have reported enhanced fusion of liposomes with a planar membrane when the planar bilayer contains a calciumbinding protein and micromolar quantities of calcium are present. However, a direct, intracellular route is not compatible with the findings of Davis, et al. [53] and Davis and Jones [136] of a vitamin D-dependent increase in calcium localizations in vesicles that gave a positive histochemical reaction for acid phosphatase (lysosomes). Although lysosomes have long been categorized as purely digestive and autolytic organelles, a

number of studies contradict this notion. The intracellular heterogeneity of the lysosomal population [89,191,192], and the selective response of the subpopulations to external stimuli [89,142, 193,194] including hormones, [89,195] suggest a multifaceted role for these organelles. More significantly, Muller et al. [179,180] have found that fusion of endocytic vesicles with lysosomes does not result in the degradation of plasma membrane-derived proteins. Instead, plasma membrane proteins are recycled to the cell surface by exocytosis [179,180]. Thus, in considering lysosomes for a role in vitamin D-dependent calcium transport, endocytosis of a calcium-binding protein of the brush border membrane need not lead to its degradation. Following fusion of such a secondary lysosome with the basal lateral membrane and concomitant release of calcium, the calcium-binding protein could be returned to the microvillar membrane by the redistribution mechanism of intestinal cells described by Quaroni et al. [131]. Moreover, released lysosomal enzymes could be retrieved by receptor-mediated pinocytosis [ 196]. A role of lysosomes in vitamin D-mediated calcium absorption could also account for observations of lipid remodeling of intestinal plasma membranes through lysosomal phospholipases C [197] and A 2 [65,198]. In other cell systems, coupling of endocytosis and exocytosis has been noted to enhance the incorporation of phosphate into phospholipids [199], stimulate the turnover of phosphatidic acid and phosphatidylinositol [200], (a phenomenon associated with calcium uptake in parotid gland [73]), as well as to require the activity of phospholipase A2 [199] the phospholipid deacyclase studied by O'Doherty [64]. Indeed, the role of phospholipase A 2 has been postulated to be integral to the process of vesicular flow more than once [89,201,202]. Despite the direct and indirect evidence for vesicular transport of calcium, very little consideration has been addressed to calcium release after fusion of the organelles with the plasma membrane. Davis et al. [53] have postulated proteolysis of the calcium-binding protein. The data of Freedman et al. [148] suggest that the relatively higher extracellular pH could effect release from vesicular binding sites. Alternatively, binding of calcium to

324 a phosphorylated protein could be postulated, with release of the salt occuring through the action of a phosphatase. The c o m b i n e d data strongly support the physiological i m p o r t a n c e of intracellular vesicles in the response o f the intestinal epithelium to v i t a m i n D. Conclusive proof that these organelles mediate v i t a m i n D - d e p e n d e n t calcium a b s o r p t i o n will require knowledge of the fate of the sequestered cation. It has not been ruled out that the striking a c c u m u l a t i o n of calcium by these vesicles occurs in order to regulate the activity of sequestered enzymes, rather t h a n for transport purposes. Some evidence does exist that vesicular calcium accumulation is involved in transport. I n studies o n the chick chorioallantoic m e m b r a n e , C o l e m a n a n d T e r e p k a [203] reported the existence of two calcium pools, one of which was exchangeable. In E P M A studies of tissue i n c u b a t e d with either calcium or strontium, it was f o u n d that the exchangeable, transport pool was located in endocytic vesicles [203]. Thus, the actions of v i t a m i n D, or its active metabolite 1,25(OH)eD 3, on the intestinal epithelial cell are pleiotropic. There are d o c u m e n t a b l e effects on the glycocalyx, brush border, and basai lateral m e m b r a n e fractions which involve changes in carbohydrate, protein, a n d lipid. I n addition, a m a j o r consequence of 1,25(OH)2D 3 is the appearance of a v i t a m i n D - i n d u c e d c a l c i u m - b i n d i n g p r o t e i n which is present in the cytosol. As yet, n o clear m e c h a n i s m of the total calcium translocation from the intestinal lumen, across the epithelial cell, to the b l o o d has been elucidated. However, current evidence supports either a model of vesicular transport or one involving protein carriers. It is anticipated that future research which focuses on the elucidation of m e m b r a n e composition, topography, and d y n a m i c properties will provide further useful i n f o r m a t i o n .

Acknowledgements The p r e p a r a t i o n of this review article was supported by U S P H S grant AM-09012-018 (A.W.N.) a n d U S P H S AM-07310 (I.N.).

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